Targeted oligonucleotide conjugates

ABSTRACT

The present invention provides improved ingress of therapeutic and other moieties into cellular targets. In accordance with preferred embodiments, complexes are provided which carry primary moieties, chiefly therapeutic moieties, to such target cells. Such complexes preferably feature cell surface receptor ligands to provide specificity. Such ligands are preferably bound to primary moieties through polyfunctional manifold compounds.

FIELD OF THE INVENTION

[0001] The present invention relates to complex compounds and methodsfor using such complex compounds. The compounds of the invention arepreferably used in methods for targeting cellular receptors thatfacilitate endocytic processes. The present invention takes advantage ofthis receptor targeting to enhance the intracellular uptake ofbiologically active compounds for therapeutic purposes.

BACKGROUND OF THE INVENTION

[0002] The use of synthetic, short, single stranded oligonucleotidesequences to inhibit gene expression has evolved to the clinical stagein humans. It has been demonstrated that incorporation of chemicallymodified nucleoside monomers into oligonucleotides can produce antisensesequences which can form more stable duplexes and can have highselectivity towards RNA (Frier et al., Nucleic Acids Research, 1997, 25,4429-4443). Two modifications that have routinely given high bindingaffinity together with high nuclease resistance are phosphorothioatesand methylphosphonates.

[0003] There are a number of desirable properties such as specificity,affinity and nuclease resistance that oligonucleotides should possess inorder to elicit good antisense activity. The ability to selectivelytarget and be taken up by diseased cells is another important propertythat is desirable in therapeutic oligonucleotides. Naturaloligonucleotides are polyanionic and are known to penetrate cells atvery low concentrations. Neutral oligonucleotides, such as themethylphosphonates, are taken up by cells at much higher concentrations.Although the processes by which antisense oligonucleotides enter thecell membrane are not well understood, there is substantial evidence fordistinct mechanisms of cell entry based on the electronic character ofthe antisense sequence.

[0004] Delivery of an antisense oligonucleotide to a specific, diseasedcell is a very important area of active research. The majority ofprojected antisense therapies are for viral infections, inflammatory andgenetic disorders, cardiovascular and autoimmune diseases andsignificantly, cancer. For example, in conventional chemotherapy,neoplasticity and virus-related infections are treated with high drugconcentrations, leading to overall high systemic toxicity. This methodof treatment does not distinguish between diseased cells and healthyones.

[0005] In the treatment of cancers, the ability of antisense agents todown-regulate or inhibit the expression of oncogenes involved intumor-transforming cells has been well documented in culture and animalmodels. For example, antisense inhibition of various expressed oncogeneshas been demonstrated in mononuclear cells (Tortora et al., Proc. Natl.Acad. Sci., 1990, 87, 705), in T-cells, in endothelial cells (Miller etal., P.O.P., Biochimie, 1985, 67, 769), in monocytes (Birchenall-Robertset al., Suppl. 1989, 13 (P.t. C), 18), in reticulocytes (Jaskulski, etal., Science 1988, 240, 1544)and in many other cell types, as generallyset forth in Table 1. TABLE 1 INHIBITION OF MAMMALIAN GENE EXPRESSIONINHIBITION OF EXPRESSION CELL TYPE T cell receptor T cellsColony-stimulating factors Endothelial cells β-Globin ReticulocytesMultiple drug resistance MCF-1 cells cAMP kinase HL-60 cells bc 1-2 L697cells c-myb Mononuclear cells c-myc T-lymphocytes Interleukins Monocytes

[0006] Virally infected cell cultures and studies in animal models havedemonstrated the great promise of antisense and other oligonucleotidetherapeutic agents. Exemplary targets from such therapy includeeukaryotic cells infected by human immunodeficiency viruses(Matsukura etal., Proc. Natl. Acad. Sci., 1987, 84, 7706; Agrawal et al., Proc. Natl.Acad. Sci., 1988, 85, 7079), by herpes simplex viruses (Smith et al.,P.O.P., Proc. Natl. Acad. Sci., 1986, 83, 2787), by influenza viruses(Zerial et al., Nucleic Acids Res., 1987, 15, 9909) and by the humancytomegalovirus (Azad et al., Antimicrob. Agents Chemother., 1993, 37,1945). Many other therapeutic targets also are amenable to suchtherapeutic protocols.

[0007] The use of non-targeted drugs, to treat disease routinely causesundesirable interactions with non-diseased cells (Sidi et al., Br. J.Haematol., 1985, 61, 125; Scharenberg et al., J. Immunol., 1988, 28, 87;Vickers et al., Nucleic Acids Res., 1991, 19, 3359; Ecker et al.,Nucleic Acids Res., 1993, 21, 1853). One example of this effect is seenwith the administration of antisense oligonucleotide in hematopoieticcell cultures that exhibit non-specific toxicity due to degradativeby-products.

[0008] Other research efforts suggest that antisense oligonucleotidespossess more side effects in both in vitro and in vivo animal models.For example, non-complementary DNA sequences have been shown tointerfere with cell proliferation and viral replication events throughunknown mechanisms of action (Kitajima ibid) . This reinforces thedesirability of oligonucleotides that are specifically targeted todiseased cells.

[0009] When phosphodiester oligonucleotides are administered to cellcultures, a concentration of typically about 1 mmol is required to seeantisense effects. This is expected since local endonucleases andexonucleases cleave these strands effectively and only 1-2% of the totaloligonucleotide concentration becomes cell-associated (Wickstorm et al.,Proc. Natl. Acad. Sci. 1988, 85, 1028; Wu-Pong et al., Pharm. Res. 1992,9, 1010). If chemically modified oligonucleotides, such as thephosphorothioates or methylphosphonates are used, the observed antisenseeffects are anywhere between 1 and 100 μM. This observed activity isprimarily due to the relatively slow cellular uptake ofoligonucleotides. There is evidence which suggests that a 80 kiloDalton(kDa) membrane receptor mediates the endocytic uptake of natural andphosphorothioate oligonucleotides in certain type of cells. Other dataquestion the existence of such a link between receptor-mediatedoligonucleotide uptake and internalization of oligonucleotides. Forexample, inhibitors of receptor-mediated endocytosis have no effect onthe amount of oligonucleotide internalized in Rauscher cells (Wu-Pong etal., Pharm. Res., 1992, 9, 1010). For uncharged methylphosphonates, itwas previously believed that internalization of such agents occurred bypassive diffusion (Miller et al., Biochemistry 1981, 20, 1874). Thesefindings were disproved by studies showing that methylphosphonates takeup to 4 days to cross phospholipid bilayers, which correlates well withthe fate of internalization of natural oligonucleotides (Akhtar et al.,Nucleic Acids Res., 1991, 19, 5551).

[0010] Increased cellular uptake of antisense oligonucleotides byadsorptive endocytosis can be obtained by liposome encapsulation. In onestudy, researchers showed that a 21-mer complementary to the 3′-tatsplice acceptor of the HIV-1 was able to markedly decrease theexpression of a p24 protein while encapsulated into a liposomecontaining diastearoylphosphatidylethanol-amine (Sullivan et al.,Antisense Res. Devel., 1992, 2, 187). Many other examples have beenreported, including pH-sensitive liposomes (Huang et al., MethodsEnzymol., 1987, 149, 88) which are well detailed in several good reviewarticles (Felgner et al., Adv. Drug Deliv. Rev., 1990, 5, 163 andFarhood et al., N.Y. Acad. Sci., 1994, 716, 23). When phosphorothioateoligonucleotides, that are complementary to the methionine initiationcodon of human intracellular adhesion molecule-1, were encapsulated, a1000-fold increase of antisense potency was seen relative to thenon-encapsulated phosphorothioate oligonucleotide (Bennett et al., Mol.Pharmacol., 1992, 41, 1023). The oligonucleotide delivery systems aregood for in vitro cell systems, but have not been shown to be widelyapplicable to in vivo studies, due to rapid liposome destabilization andnon-specific uptake by liver and spleen cells.

[0011] Other, non-specific oligonucleotide uptake enhancements attendattaching hydrophobic cholesterol (Letsinger et al., Proc. Natl. Acad.Sci., 1989, 86, 6553) type or phospholipid type molecules (Shea et al.,Nucleic Acids Res., 1990, 18, 3777) to the oligonucleotides. It has beenshown that the coupling of a single cholesterol moiety to an antisenseoligonucleotide increases cellular uptake by 15-fold (Boutorin et al.,FEBS Lett., 1989, 254, 129). When the cationic polymeric drug carrierpoly(L-lysine) is conjugated to oligonucleotide sequences, a markedincrease of non-specific oligonucleotide cellular uptake occurs(Lemaitre et al., Proc. Natl. Acad. Sci., 1987, 84, 648; Leonetti etal., Gene 1988, 72, 323; Stevenson et al., J. Gen. Virol., 1989, 70,2673). This cationic polymer has been used to deliver several types ofdrugs with cellular uptake mediated by an endocytic-type mechanism.However, the high molecular weight polylysine is cytotoxic even at lowconcentrations.

[0012] Cell surface receptors are good candidates to serve as selectivedrug targets. The presence of specific receptors implies that naturalendogenous ligands are also present. It is the complexation of theligand with the appropriate receptor that elicits a cascade of cellularevents leading to a desired function. An oligonucleotide drug linked tosuch an endogenous ligand or a synthetic ligand of equal affinitytowards the receptor in question, is considered “targeted” to thereceptor.

[0013] The potential of carbohydrate drug targeting has becomeincreasingly apparent (Shen et al., N.Y. Acad. Sci., 1987, 507, 272;Monsigny et al., N.Y. Acad. Sci., 1988, 551, 399; Karlsson et al., TIPS1991, 12, 265) as an alternate method for site-specific drug delivery.Complex carbohydrates are involved in many cellular recognitionprocesses such as adhesion between cells, adhesion of cells to theextracellular matrix, and specific recognition of cells (Ovarian eggwith sperm) by one another (Yamada, K.M., Annu., Rev. Biochem., 1983,52, 761; Edelman, G.M., Annu. Rev. Biochem., 1985, 54, 135; Hook et al.,Annu. Rev. Biochem., 1984, 53, 847; Florman, H.M., Cell, 1985, 41, 313.It is also known that the concentrations of various glycosylatedproteins that circulate in the blood are constantly regulated by cellsin various tissues. Nature controls and regulates such diverse functionswith the aid of specific proteins appearing at the surface of variouscells, which have the ability to decode the information found in complexcarbohydrate structures. These proteins are collectively called lectinsand act as receptors for carbohydrates (Goldstein et al., Nature, 1980,285, 66). Many endogenous lectins are expressed at the surface of normaland malignant cells and are involved in many poorly understoodbiological processes.

[0014] The structural information obtained from a large number ofmammalian lectins has led to their classification into several families.TABLE 2 PROPERTIES OF C-TYPE AND S-TYPE ANIMAL LECTINS PROPERTY C-TYPELECTINS S-TYPE LECTINS Ca⁺⁺⁻ dependance Yes No Solubility VariableBuffer soluble Location Extracellular Intracellular/extra State ofcysteines Disulf ides cellular Carbohydrate Different types Free thiolsspecificity Mostly β- galactosides

[0015] The C-lectins or calcium-dependent lectins possess carbohydraterecognition domains (CRDs) of the 115-134 amino acids which contain 18highly conserved and 14 invariant residues (Drickamer,K., J. Biol.Chem., 1988, 263, 9557; Drickamer, K., Curr. Opin. Struc. Biol., 1993,3, 393; Drickamer, K., Biochemical Society Transactions, 1993, 21, 456).The C-lectins are interesting from a pharmacological point of view sincethey recognize specific carbohydrates and immediately endocytose thereceptor-bound glycoprotein complex via coated pits and vesicles.

[0016] These vesicles which are also referred to as endosomes, bring thereceptor-glycoprotein complex to other cellular compartments, called thelysozomes, where protein degradation occurs (Breitfeld et al., Int. Rev.Cytol., 1985, 97, 4795). The range of C-type lectin carbohydratespecificity differs form cell to cell and from tissue to tissue.

[0017] The first membrane lectin was characterized on hepatocyte livercells (Van Den Hamer et al., J. Mol. Biol., 1970, 245, 4397). Thehepatic asialoglycoprotein receptor (ASGP-R) was isolated by Ashwell andHarford (Ashwell, G.; Herford, J., Ann. Rev. Biochem. 1982, 51, 531;Schwartz, A.L., CRC Crit. Rev. Biochem., 1984, 16, 207). These lectinsinternalized efficiently and cleared plasma levels from ceruloplasminwhich contained abnormally truncated N-oligosaccharides lacking theterminal sialic acid residues. Other artificial molecules which haveterminal galactose or N-acetylgalactosamine residues have been found tobind with high affinity to this lectin. This unique specificity betweenthe exposed galactose units and the ASGP-R suggested the design andtesting of glycotargeting systems and the use of lectins as specificdrug delivery targets. TABLE 3 MEMBRANE SPANNING C-TYPE LECTINS NAMETISSUE SUGAR SPECIFICITY ASGP-R (type II) Liver Hepatocytes Galactoseand N-acetylgalactoseamine¹ Placental Placenta Fucose and mannose² (typereceptor II) Macrophage Liver Kupffer Galactose and receptor cells typeII) N-acetyl galactoseamine Kupffer cell Liver Kupffer Galactose andfucose receptor cells (type II) IgE Fc receptor B cells Galactose⁵ (typeII) P-selectin Platelets Fucose and sialic acid(type IV) E-selectinEndothelial cells Fucose and sialic (type (type IV) IV) acid⁷ L-selectinLeukocytes Fucose and sialic acid⁸ (type IV) Mannose receptorMacrophages Mannose and fucose⁹ (type VI)

[0018] 1 Spiess et al., J. Biol. Chem., 1985, 260, 1979.

[0019] 2 Curtis et al., Proc. Natl. Acad. Sci., 1992, 89, 8356.

[0020] 3 Ii, et al., J. Biol. Chem., 1990, 265, 11295.

[0021] 4 Hoyle et al., J. Biol. Chem., 1988, 263, 7487.

[0022] 5 Kikutani et al., Cell, 1986, 47, 657.

[0023] 6 Johnston et al., Cell, 1989, 56, 1033.

[0024] 7 Bevilacqua et al., Science, 1989, 243, 1160.

[0025] 8 Laskyk et al., Cell, 1989, 56, 1045.

[0026] 9 Taylor et al., J. Biol. Chem., 1992, 267, 1719.

[0027] Many other cell lines, some summarized in Table 2, have surfacecarbohydrate-type receptors that mediate uptake of various ligands(Drickamer, K., Cell 1991, 67, 1029). Immune cells like monocytes andmacrophages possess a number of surface glycoproteins that enable themto interact with invading micro-organisms (Gordon et al., J. Cell Sci.Suppl., 1988, 9, 1). Drugs need to be carried to target cells via acarrier or high affinity ligand which is attached to the drug. Thedifferent carriers for glycotargeting can be glycoproteins orneoglycoproteins, (glycopeptides or neoglycopeptides) and asglycosylated polymers.

[0028] The in vitro glycotargeting principle is relatively simple, butits in vivo applicability is difficult. Synthetic efforts have generatedliposomes (also referred as immunoliposomes) and polylysine carriers, inwhich antibodies and some carbohydrate conjugate ligands have beencovalently attached on the outer bilayer. For example, when naturaloligonucleotides complementary to the translation initiation region ofVSV N protein mRNA were encapsulated with liposomes whose outer membranehad several H2K-specific antibodies to L929 cells, there was a markeddecrease in viral replication only within L929 infected cells (Leonettiet al., Proc. Natl. Acad. Sci., 1990, 87, 2448). Receptor-mediatedendocytic mechanisms have been exploited by attachment of cell-specificligands and antibodies to polylysine polymers. For example, c-mybantisense oligonucleotides conjugated with polylysine-folic acid (Citroet al., Br. J. Cancer 1992, 69, 463) or polylysine-transferrin (Citro etal., Proc. Natl. Acad. Sci., 1992, 89, 7031) targets were found tobetter inhibit HL-60 leukemia cell line proliferation thanoligonucleotides without conjugated carriers. Another promisingpolylysine-asialoorosomucoid carrier was conjugated withphosphorothioate oligonucleotides complementary to the polyadenylationsignal of Hepatitis B virus (Wu, G.Y.; Wu, C.H., J. Biol. Chem., 1992,267, 12436).

[0029] Methods have been previously developed that utilize conjugates toenhance transmembrane transport of exogenous molecules. Ligands thathave been used include biotin, biotin analogs, other biotinreceptor-binding ligands, folic acid, folic acid analogs, and otherfolate receptor-binding ligands. These materials and methods aredisclosed in U.S. Pat. No. 5,108,921, issued Apr. 28, 1992, entitled“Method for Enhanced Transmembrane Transport of Exogenous Molecules”,and U.S. Pat. No. 5,416,016, issued May 16, 1995, entitled “Method forEnhancing Transmembrane Transport of Exogenous Molecules”, thedisclosures of which are herein incorporated by reference.

[0030] These immunoliposomes and antibody-polymer targeting exhibited noin vivo activity. With similar drawbacks as their non-specificcounterparts, the immunoliposome-drug complexes are mostly immunogenicand are phagocytosed and eventually destroyed in the lysosomecompartments of liver and spleen cells. As for theantibody-polylysine-drug complexes, they have shown substantial in vitrocytotoxic activity (Morgan et al., J. Cell. Sci., 1988, 91, 231). Othercarriers are glycoproteins. On such large structures, a few drugmolecules can be attached. The glycoprotein-drug complexes cansubsequently be desilylated, either chemically or enzymatically, toexpose terminal galactose residues.

[0031] Glycoproteins and neoglycoproteins are recognized by lectins suchas the ASGP-R. Glycoproteins having a high degree of glycosylationheterogeneity are recognized by many other lectins making targetspecificity difficult (Spellman, N.W., Anal. Chem., 1990, 62, 1714).Neoglycoproteins having a high degree of homogeneity exhibit a higherdegree of specificity for lectins especially the ASGP-R. Manyexperimental procedures which are used to couple sugars to proteins havebeen reviewed by Michael Brinkley (Brinkley, M., Bioconjugate Chem.,1992, 3, 2). These neoglycoproteins may mimic the geometric organizationof the carbohydrate groups as in the native glycoprotein and should havepredictable lectin affinities. Successful in vitro delivery ofAZT-monophosphate, covalently attached to a human serum albumincontaining several mannose residues was achieved in human T4 lymphocytes(Molema et al., Biochem. Pharmacol., 1990, 40, 2603).

[0032] Examples of antisense oligonucleotide-neoglycoprotein complexeshave been previously reported (Bonfils et al., Nucleic Acids Res., 1992,20, 4621). The authors mannosylated bovine serum albumin and attached,covalently from the 3′-end, a natural oligonucleotide sequence. Theoligonucleotide-neoglycoprotein conjugate was internalized by mousemacrophages in 20-fold excess over the free oligonucleotide.Biotinylated oligonucleotides, were also disclosed which werenon-covalently associated with mannosylated streptavidin (Bonfils etal., Bioconjugate Chem., 1992, 3, 277). Such complexes were also betterinternalized by macrophages. Other successful examples consisted ofantisense oligonucleotides which were non-covalently associated withasialoglycoprotein-polylysine conjugates. Such oligonucleotideconjugates were found to internalize more efficiently into hepatocytes(Bunnel et al., Somatic Cell Molecular Genetics, 1992, 18, 559; Reiniset al., J. Virol. Meth., 1993, 42, 99) and into hepatitis B infectedHepG2 cells (Wu, G.Y.; Wu, C.H., J. Biol. Chem., 1992, 267, 12436).

[0033] Polymeric materials have been assessed as drug carriers and threeof them, dextrans, polyethyleneglycol (PEG) andN-(2-hydroxypropyl)methacrylamide (HMPA) co-polymers, have beensuccessfully applied in vivo (Duncan, R., Anticancer Drugs, 1992, 3,175). This research has been focused mainly at treatments for cancer andas a requisite the size of the compounds are between 30-50 kDa to avoidrenal excretion (Seymour, L.W., Crit. Rev. Ther. Drug Carrier Syst.,1992, 9, 135).

[0034] In order to examine the chemistry and related methodologiesinvolving the preparation of glycoprotein-drug and neoglycoprotein-drugglycoconjugates, certain groups have investigated the use of simplerhigh affinity ligands for specific drug delivery. Initially, severalsugars were attached to small peptides in an attempt to obtain mimics ofmultivalent N-linked oligosaccharides (Lee, R.T., Lee, Y.C.,Glycoconjugate J., 1987, 4, 317; Plank et al., Biconjugate Chem., 1992,3, 533; Haensler et al., Bioconjugate Chem., 1993, 4, 85).

[0035] Other groups investigated the use of sugar clusters lacking aprotein backbone and eventually used low molecular weight N-linkedoligosaccharides with a minimum carbohydrate population to bind withhigh affinity to lectins as the ASGP-R. Branched N-linkedoligosaccharide-drug conjugates can be used instead ofneoglycoprotein-drug complexes. The total synthesis of branched N-linkedoligosaccharides is still a difficult task, however they could beobtained by enzymatic cleavage from protein backbones (Tamura et al.,Anal. Biochem., 1994, 216, 335). This method requires expensivepurifications and only generates low quantities of chemically definedcomplex oligosaccharides. The affinity of N-linked oligosaccharideclusters towards many lectins has been demonstrated and has helpedresearchers to locate different new mammalian lectins in animals (Chiuet al., J. Biol. Chem., 1994, 269, 16195).

[0036] One process of increasing the intracellular oligonucleotideconcentration is via receptor-mediated endocytic mechanisms. This noveldrug targeting concept has been demonstrated in vitro by several groups.Oligonucleo-tides have been attached to glycoproteins, neoglycoproteinsand neoglycopolymers possessing a defined carbohydrate population which,in turn, are specifically recognized and internalized by membranelectins. To the best of our knowledge in vivo applicability ofoligonucleotide-carbohydrate conjugates has not been previouslydemonstrated.

[0037] It has also been shown in in vitro experiments that syntheticneoglycoproteins dontaining galactopyranosyl residues at non-reducingterminal positions are recognized by the ASGP-R with increasing affinityas the number of sugar residues per molecule is increased (Kawaguchi etal., J. Biol. Chem. 1981, 256, 2230).

OBJECTS OF THE INVENTION

[0038] As in apparent, there exists a need for an improved method ofselective delivery of biologically active compounds such as antisenseoligonucleotides to specific cells. This invention is directed toproviding methods to effect such delivery.

SUMMARY OF THE INVENTION

[0039] The present invention provides complexes and methods for usingsuch complexes. The complex forms are useful for enhancing theintracellular uptake of biologically active compounds (primarycompounds). The complex compounds of the invention are prepared havingthe component parts shown below:

[0040] wherein the primary moiety is a nucleotide, nucleoside,oligonucleotide or oligonucleoside; each of said linkers are,independently, bi-or trifunctional; said manifold is derivatized at aplurality of sites;each of said cell surface receptor ligands is acarbohydrate; and n is an integer from 2 to about 8.

[0041] Preferably, at least two cell surface receptors are individuallylinked by linker groups to a manifold compound which is further linkedto a primary compound. The cell surface receptor ligands impart affinityto the complexes for cells having surface receptors that recognize theselected cell surface receptor ligands. This interaction is believed totrigger endocytosis of the complex, resulting in an increased uptake bythe cell of the primary compound.

[0042] In one embodiment of the invention primary compounds are selectedto be oligonucleotides or oligonucleosides. Attachment ofoligonucleotides or oligonucleosides to a manifold compound, can beconveniently made at the 5′or 3′phosphate of the 5′or 3′terminalnucleotide or nucleoside of the oligonucleotide or oligonucleoside.Alternatively, the phosphate group can be introduced as part of thelinker group attached to the manifold moiety. Such a coupling is made byselecting the terminus of the linker to be a hydroxyl group andconverting it to a phosphoramidite. The phosphoramidite can then bereacted with an unblocked 2′, 3′or 5′hydroxyl group of anoligonucleotide or oligonucleoside.

[0043] Manifold species as used in the present invention can include awide variety of compounds that have functional groups or sites that canbe linked by linker groups to a primary compound together with a cellsurface receptor ligand, or, preferably ligands. In one embodiment apolycyclic molecule may be selected as the manifold compound, as can beillustrated for cholic acid. In other embodiments a smaller, monocyclicmanifold compound can be selected, such as phenyl or cyclohexyl.

[0044] Manifold compounds can also comprise branched chain aliphaticcompounds that have the funtionalities available for linking. Also,combinatorial chemistry techniques are known to utilize numerouscompounds that can be used as manifold compounds. Many combinatorialscaffolds are ammenable for use as manifold compounds by virtue of theirmultiple reactive sites, which can be subjected to various orthogonalprotection schemes. Preferable reactive sites include hydroxyl groups,carboxylic acid groups, amino groups and thiol groups.

[0045] Linker groups that are preferred for use in the present inventioncan be selected for a variety of chemical reasons. If the primarycompound is an oligonucleotide or oligonucleoside, a linker can beconveniently chosen having a secondary hydroxyl group and/or a primaryhydroxyl group with an additional functionality such as an amino,hydroxyl, carboxylic acid, or thiol group. The additional functionalitycan be used to attach one end of the linker group to the manifold moietyby for example an amide linkage. The secondary and or primary hydroxylgroups can be used to prepare a DMT or DMT phosphoramidite asillustrated in the Example section. This enables the attachment to anoligonucleotide or oligonucleoside to a solid support or to the 2′, 3′or5′position or a ribosyl group. This will allow variability of theplacement of the conjugated manifold compound to the primary compound.In a preferred embodiment an oligonucleotide is prepared using standardautomated solid support protocols as is well known in the art and theconjugated manifold compound is coupled as the last step to the 5′-◯position of the completed oligonucleotide or oligonucleoside. Cleavagefrom the solid support will give the complex compound.

[0046] For use in antisense and similar methodologies, oligonucleotidesand oligonucleosides of the invention preferably comprise from about 10to about 30 subunits. It is more preferred that such oligonucleosidescomprise from about 15 to about 25 subunits. As will be appreciated, asubunit is a base and sugar combination suitably bound to adjacentsubunits through, for example, a phosphorous-containing (e.g.,phosphodiester) linkage or some other linking moiety. The nucleosidesneed not be linked in any particular manner, so long as they arecovalently bound. Exemplary linkages are those between the 3′- and5′-positions or 2′-and 5′positions of adjacent nucleosides. Exemplarylinking moieties are disclosed in the following references: Beaucage, etal., Tetrahedron 1992, 48, 2223 and references cited therein; and U.S.patent applications Ser. No. 703,619, filed May 21, 1991; Ser. No.903,160, filed Jun. 24, 1992; Ser. No. 039,979, filed Mar. 20, 1993;Ser. No. 039,846, filed Mar. 30, 1993; and Ser. No. 040,933, filed Mar.31, 1993. Each of the foregoing patent applications are assigned to theassignee of this invention. The disclosure of each is incorporatedherein by reference.

[0047] In other embodiments of the invention, primary compounds compriseoligonucleotides or oligonucleosides attached through a linking moietyto the manifold moiety such as by a free 2′-, 3′-, or 5′-hydroxyl group.Such attachments are prepared by, for example, reacting nucleosidesbearing at least one free 2′-, 3′-, or 5′-hydroxyl group under basicconditions with a linking moiety having a leaving group such as aterminal L-(CH₂)-etc. function, where L is a leaving group. Displacementof the leaving group through nucleophilic attack of (here) an oxygenanion produces the desired derivative. Leaving groups according to theinvention include but are not limited to halogen, alkylsulfonyl,substituted alkylsul-fonyl, arylsulfonyl, substituted arylsulfonyl,hetercyclcosulfonyl or trichloroacetimidate. A more preferred groupincludes chloro, fluoro, bromo, iodo,p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl(mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl,trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy,2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl, and2,4,6-trichlorophenyl, with bromo being preferred.

[0048] Suitably protected nucleosides can be assembled into anoligonucleosides according to many known techniques. See, e.g.,Beaucage, et al., Tetrahedron 1992, 48, 2223.

[0049] A wide variety of linker groups are known in the art that will beusefulin the attachment of primary compounds to manifold compounds. Manyof these are also useful for the attachment of cell surface receptorligands to the manifold compound. A review of many of the useful linkergroups can be found in Antisense Research and Applications, S. T. Crookeand B. Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350. Adisulfide linkage has been used to link the 3′terminus of anoligonucleotide to a peptide (Corey, et al., Science 1987, 238, 1401;Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; and Corey, etal., J. Am. Chem. Soc. 1989, 111, 8524). Nelson, et al., Nuc. Acids Res.1989, 17, 7187 describe a linking reagent for attaching biotin to the3′-terminus of an oligonucleotide. This reagent,N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available fromClontech Laboratories (Palo Alto, Calif.) under the name 3′-Amine on. Itis also commercially available under the name 3′-Amino-Modifier reagentfrom Glen Research Corporation (Sterling, Va.). This reagent was alsoutilized to link a peptide to an oligonucleotide as reported by Judy, etal., Tetrahedron Letters 1991, 32, 879. -A similar commercial reagent(actually a series of such linkers having various lengths ofpolymethylene connectors) for linking to the 5′-terminus of anoligonucleotide is 5′-Amino-Modifier C6. These reagents are availablefrom Glen Research Corporation (Sterling, Va.). These compounds orsimilar ones were utilized by Krieg, et al., Antisense Research andDevelopment 1991, 1, 161 to link fluorescein to the 5′-terminus of anoligonucleotide. Other compounds such as acridine have been attached tothe 3′-terminal phosphate group of an oligonucleotide via apolymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci. USA1984, 81, 3297).

[0050] Oligonucleotides have been prepared on solid support and thenlinked to a peptide via the 3′hydroxyl group of the 3′terminalnucleotide (Haralambidis, et al., Tetrahedron Letters 1987, 28, 5199).An Aminolink 2 (Applied Biosystems, Foster City, Calif.) has also beenattached to the 5′terminal phosphate of an oligonucleotide (Chollet,Nucleosides & Nucleotides 1990, 9, 957). This group also used thebifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) tolink an interleukin protein to an oligonucleotide.

[0051] In another embodiment of the invention, linker moieties are usedto attach manifold groups to the 5 position of a pyrimidine (Dreyer, etal., Proc. Natl. Acad. Sci. USA 1985, 82, 968). Fluorescein has beenlinked to an oligonucleotide in the this manner (Haralambidis, et al.,Nucleic Acid Research 1987, 15, 4857) and biotin (PCT applicationPCT/US/02198). Fluorescein, biotin and pyrene were also linked in thesame manner as reported by Telser, et al., J. Am. Chem. Soc. 1989, 111,6966. A commercial reagent, Amino-Modifier-dT, from Glen ResearchCorporation (Sterling, Va.) can be utilized to introduce pyrimidinenucleotides bearing similar linking groups into oligonucleotides.

[0052] Cholic acid linked to EDTA for use in radioscintigraphic imagingstudies was reported by Betebenner, et.al., Bioconjugate Chem. 1991, 2,117.

[0053] In a preferred embodiment of the present invention, novel complexcompounds are prepared having oligonucleotide conjugates that are usefulfor oligonucleotide antisense drug targeting of, for example, thecarbohydrate recognition domains (CRD) found on theasiologlycoprotein-receptor (ASGP-R). These complex compounds wereprepared according to the principles which govern the specificity of the{ligand-[ASGP-R]} complex. Simple carbohydrates and Glycoconjugateshaving only one linked saccharide moiety show a slight affinity for thereceptor (Lee et al., Biol. Chem., 1983, 258, 199). For instance,glycoconjugates having monovalent ligands such as galactose, lactose ormonoantennary galactosides (one carbohydrate group attached via alinkage to the scaffold) bind to this ASGP-R with a millimolardissociation constant. When binary oligo-saccharides (two carbohydrategroups each attached via a linkage to the scaffold) are used, thedissociation constants are in the micromolar range. This translates to athree order of magnitude higher affinity. When trinary oligosaccharides(three carbohydrate groups each attached via a linkage to the scaffold)are tested, the dissociation constants are in the nanomolar range.

[0054] Based on dissociation constants, e.g. higher for 3 carbohydrategroups, trinary oligosaccharides were preferably synthesized, eachhaving three carbohydrate groups independently linked to a scaffoldwhich was further linked to an oligonucleotide. The resulting lowmolecular weight oligonucleotide conjugates were easily amenable toautomated DNA synthesis methodology. The oligonucleotide conjugates eachconsist of at least four distinct moieties, scaffold, carbohydrateattaching linker, oligonucleotide attaching linker, and carbohydrate.

[0055] Initially, cholic acid was chosen as a scaffold. Cholic acid waschosen because it is a natural product in mammalian systems, does notform a toxic metabolite and because it is commercially available at lowcost. Another reason for choosing cholic acid was that this steroidalscaffold would be a good anchor for linked carbohydrates, separating thepoints of attachment and reducing any steric interference between them.Increasing the distance between points of attachment of the linkedcarbohydrates would increase the degree of freedom and reduce the lengthrequirement of the linker to obtain high affinity with the receptor.

[0056] Galactose and lactose were initially chosen as carbohydratemoieties since they are recognized by the carbohydrate recognitiondomains (CRD) found on the asiologlycoprotein-receptor (ASGP-R).

[0057] Aminocaproate, derived from commercially availableN-Fmoc-ε-aminocaproic acid, was chosen as the carbohydrate linker andits length was based on previously reported experimental evidence(Biessen et al., J. Med.Chem., 1995, 38, 1538). The previously reportedresults indicated that cluster galactosides between 10 and 20 Å inlength were high affinity substrates for the hepatic ASGRP-R. The lengthof the linking group was initially chosen to be eight atoms long becausein conjunction with the larger area scaffold being used the result maybe better positioning of the carbohydrate components towards the CRD'sof ASGP-R.

[0058] After the cholic acid scaffold has been linked to each of thecarbohydrates and a linker group is deblocked and ready for attachmentan oligonucleotide is coupled. The preferred method of coupling of theconjugate to an oligonucleotide is to perform the coupling while thefull length oligonucleotide is bound to solid support. The conjugate iscoupled to the oligonucleotide followed by cleavage of the final productfrom the solid support. This cleavage step also removes acetylprotecting groups present on any hydroxyl groups that were previouslyprotected especially on any saccharide moieties.

[0059] The oligonucleotide analog is tested for affinity towards theASGP-R expressed on several cells. The binding of the oligonucleotideconjugate to the ASGP-R should initiate the internalization process andincrease the intracellular concentration of the selectedoligonucleotide.

[0060] It will be appreciated that modified nucleotide moieties may alsobe useful in connection with embodiments of this invention. Thus, a widevariety of chemical modifications may be employed throughout the nucleicacids. Thus modifications of pyrimidine or purine bases, substitutionsat the 2′position, alteration of inter-nucleoside linkages, carbohydratering substitutions and positional variations may all be employed.

[0061] Teachings regarding the synthesis of modified oligonucleotidesmay be found in the following U.S. patents or pending patentapplications, each of which is commonly assigned with this application:U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for thepreparation of oligonucleotides having chiral phosphorus linkages; U.S.Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides havingmodified backbones; U.S. Pat. No. 5,386,023, drawn to backbone modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-◯-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. Nos. 5,223,168, issued Jun. 29, 1993,and 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs;U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone modifiedoligonucleotide analogs; and U.S. patent application Ser. No.08/383,666, filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255, drawn to,inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.

[0062] It is to be understood that all modifications to nucleotides,nucleosides at oligonomers thereof are encompassed within theirrespective definitions.

[0063] Antisense oligonucleotides are conversely synthesized usingautomated DNA synthetic methodology. Therefore, small glycotargetingsystems, which can be incorporated into the last cycle of an automatedDNA synthesis, are preferred.

EXAMPLE 1

[0064] Synthesis of 2,3,4,5-aceto-1-bromo-α-D-galactose

[0065] The title compound was synthesized using slightly modified, knownprocedures to generate the acetobromo-galactose (Methods in CarbohydrateChemistry, Wistler and Wollfrom, Eds, Academic Press; New York, 1962;Vol. 1-6). Dry D-galactose was reacted with acetic anhydride (20 eq.) inthe presence of a catalytic amount of DMAP (0.1 eq.) in dry pyridine at0° C. After 1 hour at 0° C., the reaction mixture was allowed to warm toroom temperature. After 6 hours had passed the thick solution was pouredinto a rapidly stirred solution of ice-water (500 mL). A stickyprecipitate developed and was extracted with ethyl acetate.1,2,3,4,5-pentaacetyl-α-D-galactose was obtained as a slightly yellowoil in 89% yield.

[0066] The 1,2,3,4,5-pentaacetyl-α-D-galactose was used without furtherpurification in the next step by treatment with HBr (5 molar eq., 30%solution in glacial acetic acid). After one hour, the HBr and the aceticacid was removed, giving rise to the title α-bromogalactoside as a thickbrown oil in 94% yield which was used for the next step without furtherpurification.

EXAMPLE 2

[0067] Synthesis of α-D-lactosyl Acid Bromide

[0068] Following the procedures illustrated for Example 1 above, dryD-lactose was transformed to octaacetylated D-lactose, which wasobtained as a 20:80 mixture of α/β-anomers (α-anomer: doublet, 6.19 ppm,JH_(H1′−H2′)=3.7 Hz, H1′; β-anomer: doublet, 5.1 ppm, JH_(H1′−H2′)=8.4Hz, H1′) in 76% yield. The octaacetate was used without furtherpurification by treatment with a solution of HBr (5 molar eq., 30%solution in glacial acetic acid). After the lactosyl compound dissolved(˜4-5 min.), the HBr and the acetic acid were quickly removed to avoidhydrolysis of the β(1-4) -◯-glycosidic linkage. The title compound wasobtained as a thick brown oil in 97% yield. The title compound was usedwithout further purification in the next step.

EXAMPLE 3

[0069] Synthesis of Peracetylated-β-azidogalactose

[0070] 2,3,4,5-Aceto-1-bromo-α-D-galactose was reacted under phasetransfer catalysis (PTC) conditions previously reported (Tropper et al.,Ph.D Thesis, University of Ottawa, 1992.) with 5 eq. of sodium azide inthe presence of tetrabutylammonium hydrogen sulfate (PTS) (1 eq.) in a1:1 mixture of CH₂Cl₂ and NaHCO₃ (saturated solution). After 3 hours,under vigorous stirring, the title compound was obtained as a paleyellow solid in 97% yield. The characteristic ¹H-NMR [anomeric α-H; 4.56ppm (³JH_(H1′−H2′)=8.6 Hz)] and ¹³C-NMR [anomeric C-1′; 88.29 ppm] dataconfirmed the structure and was in agreement with published data. Thisreaction occurred with complete inversion (³JH_(H1′−H2′)=8.6 Hzconfirmed the 1,2-trans-β-D-anomeric configuration) and thereforeestablished the desired stereochemistry.

EXAMPLE 4

[0071] Peracetylated-β-azidolactose

[0072] Following the procedures illustrated for Example 1 above,α-D-lactosyl acid bromide was converted to the β-azidolactose. The titlecompound was obtained as a pale orange oil in 92% yield. Thecharacteristic ¹H-NMR [glucose anomeric α-H; 4.6 ppm (³J_(H1′−H2′)=8.8Hz)] and ¹³C-NMR [glucose anomeric C-1′; 88.30 ppm] data confirmed thestructure and was in agreement with published data (Tropper et al.,Synthesis, 1992, 7, 618). The PTC reaction occurred with completeinversion as observed for the peracetylated-β-azidogalactose.

EXAMPLE 5

[0073] Synthesis of Peracetylated-β-aminogalactose

[0074] Hydrogenation of peracetylated-β-azidogalactose in the presenceof a large excess of Pd (10% on charcoal in wet methanol) at 40 p.s.i.for 1 hour gave peracetylated-β-aminogalactoside as a pale yellow oil inquantitative yield. The characteristic ¹H-NMR data (the clean doublet ofthe anomeric proton in the azide shifted from 4.6 ppm to a multiplet at5.37-5.39 ppm in amine, indicating the absence of the anomeric azidegroup) and the mass spectrum analysis (CI-NH3: m/e 347 [M]⁺) confirmedthe structure.

EXAMPLE 6

[0075] Synthesis of peracetylated-β-aminolactose

[0076] As per the procedures illustrated in Example 5,peracetylated-β-azidolactose was hydrogenated under similar reactionconditions. The β-azide was hydrogenated in the presence of Pd (10% oncharcoal in wet methanol). After a simple celite filtration, the titlecompound was obtained in quantitative yield as a pale yellow oil. Thecharacteristic ¹³C-NMR data (the galactose anomeric carbon was at 101.14ppm and the glucose anomeric carbon shifted from 87.71 ppm in azide 4 ato 84.48 ppm) and the mass spectrum analysis (FAB-NBA: m/e 636 [M+H]⁺)confirmed the structure.

[0077] The reduction reactions of Example 5 and 6 were adapted fromTropper ibid. In this previously reported procedure stoichiometricamounts of catalyst to glycosyl azides was used. This procedure wasfound to be quite dangerous when synthesizing on large scales so theamount of Pd was reduced to 0.2 to 0.4 eq. of Pd on carbon (10%). Thisamount was sufficient to reduce the glycosyl azides without affectingthe acetates (no deacetylated galactosamines or lactosamines weredetected by ¹H-NMR or ¹³C-NMR on the crude mixtures).

[0078] Due to the instability of both glycosylamines, no purificationwas performed prior to coupling with the linkers.

EXAMPLE 7

[0079] Attachment of N-Fmoc (9-fluorenylmethoxycarbonyl) protectedε-aminocaproic acid linker to peracetylated-β-aminogalactose

[0080] N-Fmoc-ε-aminocaproic acid (Scheme 8) is treated with◯-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexa-fluorophosphate(HBTU, 1 eq.) in the presence of 1-hydroxybenzotriazole (HOBt) (1 eq.)and N-methylmorpholine (NMM) (1.5 eq.) in dry DMF. After 35 min. atr.t., peracetylated-β-aminogalactose was added and the reaction wasstirred for an additional 18 hours. The reaction mixture is concentratedand purified by chromatography to give the galactosylamide in a 45%yield. In this reaction, the anionic form of the acid attacks the HBTUforming the acyloxyphosphonium salt. This reactive salt is attacked byeither ionized HOBt or ionized acid, forming the benzotri-azolyl activeester and symmetrical anhydride, respectively. The glycosylamine reactswith either of the reactive intermediates to yield the desired product.

[0081] The amide NH appeared as sharp doublet at 6.24 ppm (in CDCl₃) andcoupled strongly (from 2D-COSY) to the anomeric multiplet at 5.18-5.27ppm (3JNH-H1′=9.15 Hz). From ¹³C-NMR spectrum, the C-1′anomeric carbonwas at 78.5 ppm. All the NMR data and the mass spectrum (FAB-NBA: m/e683 [M+H])⁺) confirmed the desired structure.

[0082] Other attempts to increase the coupling efficiency wereinvestigated by using the BOP reagent instead of HBTU.N-Fmoc-ε-aminocaproic acid was reacted with BOP (1 eq.) reagent in thepresence of HOBt (1 eq.) and NMM (1.5 eq.) in dry DMF. After 1 hour atr.t., the galactosylamine was added and 18 hours later thegalactosylamide was obtained in a 51% yield after column chromatography.

[0083] The BOP method gave comparable yields and was the preferredreagent being approximately three times cheaper than HBTU. Triethylamine(TEA) was used instead of NMM since the latter is used in peptidecoupling methodology as a racemization suppressant. In the synthesis,there is no racemization and the use of triethylamine gave comparableresults.

EXAMPLE 8

[0084] Attachment of N-Fmoc Protected Linker toPeracetylated-β-aminolactose

[0085] The attachment of an N-Fmoc protected linker toperacetylated-β-aminolactose is accomplished following the procedure ofExample 7. N-Fmoc-ε-aminocaproic acid was treated with BOP reagent (1eq.) in the presence of HOBt (1 eq.) and triethylamine (1.5 eq.) in dryDMF. After 1 hour at r.t., peracetylated-β-aminogalactose was added andthe reaction was stirred for an additional 24 hours. Following apreviously reported procedure (Coste et al., J. Org. Chem., 1994, 59,2437) a 10% citric acid solution was used instead of a saturatedsolution of NH₄Cl to efficiently extract the BOP and other by-productsfrom the organic layer of the BOP coupling reaction. The stronger acidicsolution extracted most of the BOP and the trace amounts of the activeester and the symmetric anhydride intermediates, which greatlysimplified the chromatographic purification. The lactosylamide productwas obtained after column chromatography in 51% yield. From the ¹H-NMRspectrum, the amide NH at 6.64 ppm appeared as a doublet(³J_(NH-H1′)=9.3 Hz). The glucose H1′overlapped with the H3′glucose anda multiplet was observed between 5.11 and 5.21 ppm. The glucose H2′was aclean apparent triplet (J=9.8 Hz) which confirmed the β-amide linkagethrough the 1,2-trans arrangement between H2′-H2′and between H2′-H3′.The galactose and glucose anomeric carbons were at 100.8 and 77.8 ppm,respectively. From FAB-MS, the pseudomolecular ion at 971 ([M+H]⁺) andsome characteristic fragments (749 [M+H-Fmoc]⁺and 619[M+H-{Fmoc-NH-CH₂(CH₂)₄-C(O)NH}]⁺) were in agreement with the assignedstructure.

EXAMPLE 9

[0086] Fmoc cleavage ofPeracylated-β-N-[ε-(N-Fmoc)aminocaproic]-aminogalactose with Piperidineand TBAF

[0087] The peracylated-β-N-[ε-(N-Fmoc)aminocaproic]amino-galactose(product from Example 7) was treated with tetrabutylammonium fluoride(TBAF) (1.2 eq., from a 0.1 M stock solution) (see Ueki et al.,Tetrahedron Lett., 1997, 26, 560). After 2 hours at room temperature,the reaction mixture was diluted with ethyl acetate and thoroughlywashed with water. After chromatographic purification,peracylated-β-N-(ε-aminocaproic) aminogalactose was obtained in 29%yield. Characteristic ¹H-NMR data (sharp singlet at 2.11 ppmcorresponding to the free amine NH due to fast exchange from hydrogenbonding and loss of all Fmoc proton signals) and mass spectrometry(FAB-NBA m/e: 461 [M+H]^(+,) 100% relative intensity) confirmed theassigned structure.

EXAMPLE 10

[0088] Fmoc Cleavage ofPeracylated-β-N-[ε-(N-Fmoc)aminocaproic]-aminolactose with Piperidineand TBAF

[0089] The peracylated-β-N-[β-(N-Fmoc)aminocaproic] amino-lactose(Example 8) was treated with TBAF (1.2 eq.) in dry DMF for 3 hours atroom temperature. The free amine product was obtained afterchromatography in a 55% yield. Characteristic ¹H-NMR data [a sharpsinglet at 2.12 ppm, overlapping with the α-methylene protons to theamide linkage (lac-NHC(O)CH₂-), corresponded to the free amine NHD andthe loss of all Fmoc proton signals] and mass spectrometry (FAB-NBA m/e:749 [M+H]⁺) confirmed the assigned structure.

EXAMPLE 11

[0090] Coupling of Glycosylamines with the N-carbobenzyloxy (Cbz)Protected Linkers

[0091] Commercially available N-Cbz-ε-aminocaproic acid was treated withBOP reagent (1 eq.) in the presence of HOBt (1 eq.) and triethylamine(1.6 eq.) in dry DMF for 30 minutes. Peracetylated-β-aminogalactose(Example 5) was added and stirring was maintained for an additional 48hours. The organic layer was washed with a 10% citric acid solution toremove most of the BOP and trace amounts of reactive intermediates. Thecoupled galactosylamide having the Cbz protecting group on the e-aminoend of the amino caproic acid linker was obtained after chromatographyin a 60% yield. From ¹H-NMR spectrum the amide NH appeared as a sharpdoublet at 6.43 ppm (³J_(NH-H1′)=9.3 Hz). The anomeric proton appearedas a doublet of doublets at 5.20 ppm (³JH_(H1′-NH)=9.3 Hz,³J_(J1′-H2′)=8.7 Hz) which confirmed the 1,2-trans arrangement ofH1′-H2′and the β N-glycosidic bond. The anomeric carbon was at 78.4 ppmin the ¹³C-NMR spectrum and the mass spectra [Electrospray MS m/z: 617.4[M+Na]^(+,) 100; and high resolution (FAB-NBA) m/e: 595 [M+H]⁺, calc.for C₂₈H₃₉N₂O_(12,) 595.2424; found 595.2503] confirmed the assignedstructure.

EXAMPLE 12

[0092] Coupling of Peracetylated-β-aminolactose withN-Cbs-ε-aminocaproic acid

[0093] The ε-aminoCbz protected linked peracylated-β-aminolactose wasprepared following the procedure illustrated in Example 11. The productwas obtained in a 63% yield after column chromatography. From the ¹H-NMRspectrum the amide NH appeared as a sharp doublet at 6.14 ppm (³J_(NH-H1′(Glu))=9.3 Hz) and the anomeric glucose proton appeared at 5.18ppm as a doublet of doublets (3J_(H1′-H2′)=9.5 Hz, ³J_(H1′-NH)=9.3 Hz)which confirmed the β-N-linkage. The galactose H1 appeared as a broaddoublet at 4.43 ppm (³J_(H′(gal)-H2′)=7.8 Hz). Mass spectrometry[Electrospray MS m/z: 905.5 [M+Na]^(+,) 85.2 and 883.5 [M+H]^(+,) 100;FAB-NBA m/e: 595 [M+H]⁺, calc. for C₄₀H₅₅N₂O_(20,) 883.3191; found883.3348] confirmed the assigned structure.

EXAMPLE 13

[0094] Cleavage of the Cbz ofPeracylated-β-N-[ε-(N-Cbz)aminocaproic]aminogalactose

[0095] Peracylated-β-N-[ε-(N-Cbz)aminocaproic]amino-galactose wasdissolved in ethyl acetate/water/acetic acid solvent together with anequivalent weight of palladium (10% on activated charcoal as catalyst).The mixture was agitated for 1 hour at a pressure of 40 p.s.i. hydrogen.The ¹H-NMR spectrum was very clean with a characteristic sharp singletat 2.31 ppm corresponding to the free amine. All the starting materialwas consumed and no Cbz proton signals were observed.

EXAMPLE 14

[0096] Cleavage of the Cbz of Peracylated-β-N-[ε-(N-Cbz)aminocaproic]Aminolactose Hydrogenolysis

[0097] The cleavage of the Cbz group fromperacylated-β-N-[ε-(N-Cbz)aminocaproic]aminolactose was performedfollowing the procedures of Example 13. The ¹H-NMR spectrum was veryclean as observed in the galactosyl series of Example 13.

EXAMPLE 15

[0098] Synthesis of Tetrahydroxycholane, Cholane-type Anchoring Backbone

[0099] Cholic acid was reacted with borane (4 eq.) THF complex (BH₃THF)in THF at 0° C. Recrystallization of the crude material from isopropanolgave the tetrahydroxycholane in a 75% yield. The m.p. was found to be224-225° C. The methylene protons at position 24 (—CH₂OH) appeared as abroad triplet at 3.50 ppm in the ¹H-NMR spectrum. The carboxylate carbonof cholic acid, usually found at ˜170 ppm, was absent in the ¹³C-NMRspectrum of the product. The NMR data and the mass spectrum(FAB-glycerol m/e 789 [2M+H]⁺and 395 [M+H]⁺) confirmed the expectedstructure.

EXAMPLE 16

[0100] Synthesis of the t-butyldiphenylsilyl (TBDPS) Ether ofTetrahydroxycholane

[0101] Tetrahydroxycholane (Example 15) was reacted with TBDPSC1 (1 eq.)in the presence of imidazole (2 eq.) in dry DMF at 0° C. Afterpurification by chromatography the TBDPS product was obtained in an 89%yield. From the ¹H-NMR spectrum in CDCl₃, the methylene protons atposition 24 (—CH₂OSi—) appeared as a sharp triplet at 3.60 ppm (J=6.0Hz). A sharp singlet at 1.01 ppm Corresponded to the tert-butyl groupand the phenyl protons appeared between 7.33 and 7.68 ppm. The NMR dataand the mass spectrum (FAB-NBA m/e 655 [M+Na]⁺) confirmed the assignedstructure.

EXAMPLE 17

[0102] Succinimide Activation of TBDPS Ether of Tetrahydroxycholane,Synthesis of the Active Ester

[0103] The TBDPS ether of tetrahydroxycholane (Example 16) was reactedwith triphosgene (2 eq.) in dry pyridine for 15 minutes with thetemperature maintained at 0° C. N-hydroxysuccinimide (NHS) (10 eq.) wasadded and the mixture became cloudy and thickened. Within 10 minutes ofthe addition of the NHS the mixture started to become light pink and thecolor intensified slowly over the next 20-25 minutes to light red. After30 minutes the reaction mixture was poured into cold water and theactive ester precipitated out of the solution. Recrystallization from95% methanol gave a yield of 76% of the active ester. In the ¹H-NMRspectrum (FIG. 19 +L), the 3-,7-and 12-α-CH protons appeared at4.56-4.62 (m), 4.87-4.88 (m) and 5.04 (s) ppm. The downfield shift ofall the α-CH and the multiplet at 2.75-2.83 ppm, corresponding to the 12succimidyl methylene protons [-(O)C-CH₂CH₂C(O)-] confirmed the trisactivation. The NMR data and the mass spectrum (electrospray MS m/z:1078.6 [M+Na]^(+;) 1056.5 [M+H]⁺) confirmed the structure.

EXAMPLES 18-24

[0104] Preparation of Tris Peracylated Galactose and Tris PeracylatedLactose Conjugates of Formula:

Example # R Q 18 peracylated galactose TBDPS 19 peracylated lactoseTBDPS 20 peracylated galactose OH 21 peracylated lactose OH 22-24peracylated lactose DMT 25 peracylated lactose OH

EXAMPLE 18

[0105] Tris coupling of peracylated-β-N-(ε-aminocaproic)amino-peracylated galactose to the NHS active ester

[0106] Active ester (500 mg) was reacted withperacylated-β-N-(ε-aminocaproic) aminoperacylated galactose in dry DMF.After 15 hours at r.t., the reaction mixture was poured into ice coldwater and the resulting precipitate was collected and dried underreduced pressure for 24 hours. The 960 mg of product, collected fromrunning the above reaction sequence twice and combining the products,was dissolved in dry pyridine containing acetic anhydride (20 eq.). Themixture was allowed to stir for twelve hours and then was poured into200 mL of ice water. The resulting pale yellow precipitate was isolatedand dried to give a ˜90% yield. Further purification was not needed asonly one spot was obtained by TLC with an R_(f)=0.4 in 5% MeOH/CH₂Cl₂.The ¹H-NMR spectrum was quite complex. The compound was very soluble inCDCl₃ and CD₂Cl₂, but strangely yielded broad and undefined protonresonances. We do not understand the factors involved. One can speculatethat such molecules fold, interact and relax differently from non-polarto polar solvents. Only DMSO-d₆ could be used to obtain high resolutionspectrum. The three anomeric protons appeared at 5.31 ppm as an apparenttriplet. Although a J value can be extracted (J=9.3 Hz), it ismisleading to use this coupling constant since three triplets aresuperimposed. At 2.85 to 3.01 ppm, a complex set of multipletsintegrated for 6H and corresponded to the 3 [—CH₂—NH—C(O)O—] protons. Inthe acetyl CH₃ region between 1.92 and 2.07 ppm, 5 singlets and onemultiplet integrated for 36H and correlated well with trisubstitution.Both the NMR data and the electrospray mass spectra {(iPrOH/DMF/TFA)m/z: 2092.4 [M+H]^(+,) 1588.1 [M+H—C₂₁H₃₁N₂O_(12]) ^(+,) 1083.8[M+H—C₄₂H₆₂N₄O₂₄]⁺and 461 [M+H—C₈₃H₁₁₉N₄O₂₇Si]⁺; (DMF/TFA/AcOH) m/z:2092.4 [M+H]⁺; (DMF/CsI) m/z: 2224.2 [M+Cs]²⁺} confirmed the assignedstructure.

EXAMPLE 19

[0107] Tris Coupling ofPeracylated-β-N-(ε-aminocaproic)amino-peracylated Lactose to the NHSActive Ester, Tris-coupled Peracylated Galactose Conjugate

[0108] The NHS active ester was tris coupled toperacylated-β-N-(ε-aminocaproic)aminoperacylated galactose (Example 14)following the procedures illustrated in Example 18. NHS active ester wasreacted with lactosylamine (3.3 eq.) in dry DMF at room temperature.After 18 hours, the reaction mixture was poured into ice water and thetrisubstituted lactosyl glycoconjugate was obtained in 87% yield. Themono-deacetylated material (less than 10% by electrospray mass spectra)was reacetylated by treatment with acetic anhydride.

[0109] The ¹H-NMR spectrum of the title compound was complex. Three setsof complex multiplets at 5.10-5.28 ppm integrating for 12H correspondedto three glucose anomeric protons, three H4′(peracylated galactose),three H3′(glucose) and three H3′(peracylated galactose), as deduced from2D-COSY spectra. The three H1′(glucose) overlapped and appeared as oneapparent triplet with ³J-coupling values (9.0 and 12.0 Hz) which are notaccurate for any of the three anomers. Between 0.68 and 2.08 ppm, acomplex area was found to integrate to ˜170H. The integration accountedfor all the steran H and —CH₃ groups, 9 —CH₂—caproic groups, 3—CH₂—groups adjacent to the amide linkage, and 63H for the 21 acetyl—CH₃ groups. From ¹³C-NMR, the peracylated galactose anomeric carbon wasat 99.78 ppm and the glucose anomeric carbon at 76.54 ppm. The NMR dataand the mass spectrum [electrospray (DMF) m/z: 2978.2 [M+Na]^(+;) 2186.2[M+Na—C₃₃H₄₇N₂O₂₀]^(+] confirmed the assigned structure.)

EXAMPLE 20

[0110] Fluoride-promoted Desilylation of Tris-coupled PeracylatedGalactose Conjugate

[0111] The peracylated galactose conjugate (title compound of Example18) was desilylated by treatment with TBAF (25 eq.) in the presence ofAcOH (12 eq.) in dry THF. After 1 hour at room temperature additionalTBAF (13 eq.) was added and the progress of the reaction was closelymonitored. After 30 minutes from the second addition of TBAF, there wasstill starting material left and a third spot (below the product)appeared. The reaction mixture was immediately poured into ice water toavoid further deacetylation of the product. A sticky precipitatedeveloped and was extracted in ethyl acetate.

[0112] Several water washes were performed to remove the excess TBAF andafter silica gel column chromatography using 2-2.5% MeOH in CH₂Cl₂,three fractions were obtained. The first consisted of some startingmaterial which overlapped with the desilylated product (two spots; topR_(f)=0.28, bottom R_(f)=0.21 in 5% MeOH/CH₂Cl_(2;) 19%) . The middlefractions consisted essentially of desilylated tris-coupled peracylatedgalactose conjugate (one middle spot; R_(f)=0.21 in 5% MeOH/CH₂Cl_(2;)55%) and the last fraction was a mixture of desilylated product anddeacetylated by-products (˜15%). The TBAF method was sufficientlyacceptable for the synthesis of 500 mg of pure desilylated tris-coupledperacylated galactose conjugate, required for biological testing.

[0113] The ¹H-NMR in DMSO-d₆ (middle spot, 23) was very sharp andseveral assignments were made (FIG. 22 +L). Three carbamate NH'sappeared as broad multiplets between 6.60 and 6.84 ppm. The threeH1′protons appeared at 5.32 ppm as a sharp apparent triplet. TheH2′protons appeared between 4.97 and 5.02 ppm as a multiplet withtriplet-like character.

[0114] The 500 MHZ ¹H-NMR and 2D-COSY spectrum of the desilylatedtris-coupled peracylated galactose conjugate showed 24-OH groupsappeared between 3.30 and 3.40 ppm as a very intense broad singlet whichoverlapped with residual water. The t-butyl and phenyl group H's wereabsent. The above NMR data and the electrospray mass analysis[(DMF/AcOH) m/z: 1876.7 ([M+H+Na]^(+,) 100), 1854.4 ([M+H]^(+,) 5.15),1371.8 ([M+H—1—C₂₁H₃₁N₂O₁₂+Na]^(+,) 9.82), 1349.8([M+H-1-C₂₁H₃₁N₂O_(12]) ^(+, 9.82)), 527.2 ([M+H—C₆₆H₁₀₁N₄O₂₅+Na]^(+,) 5.73), 503.4 ( [M+H—C66H₁₀₁N₄O₂₅]^(+,)2.54)] confirmed the structure assigned for the desilylated tris-coupledperacylated galactose conjugate.

EXAMPLE 21

[0115] Fluoride-promoted Desilylation of Tris-coupled PeracylatedLactose Conjugate

[0116] The tris-conjugated peracylated lactose conjugate (Example 19)was desilylated using the procedures of Example 20. The desilylatedtris-coupled peracylated lactose conjugate was obtained in 37% yield.Some product overlapped with 20% of starting material and withappreciably more deacetylated by-products (˜25%). The ¹H-NMR of theproduct (middle spot) in DMSO-d₆ was very sharp and several protons wereassigned in conjunction with the 2D-COSY spectrum.

[0117] The 500 MHZ ¹H-NMR and 2D-COSY spectrum showed three carbamateNH's that appeared as broad multiplets between 6.59 and 6.84 ppm.Between 5.09 and 5.33 ppm, three complex multiplets integrated for 12protons. After evaluation of the 2D-COSY spectra for this area thesignals were assigned to three sets of H1′[glucose], H4′[peracylatedgalactose], H3′[glucose] and H3′[peracylated galactose]. TheH2′peracylated galactose proton appeared as an apparent doublet ofdoublets at 4.83 ppm. The 24-OH group appeared between 3.28 and 3.33 ppmas a very intense broad singlet which overlapped with residual water.The t-butyl and phenyl group H's were absent. From ¹³C-NMR and2D-Heteronuclear Multiple Quantum Coherent (HMQC) spectra, many carbonswere assigned within the carbohydrate and linker regions. The threeanomeric glucose carbons were at 76.50, 76.52 and 76.55 ppm. Theperacylated lactose anomeric carbons were at 99.80, 99.77 and 99.70 ppm.The 24-CH₂OH carbon was at 61.17 ppm. The above NMR data and theelectrospray mass analysis [(DMF/AcOH) m/z: 2740.1 ([M+Na]^(+,) 100.0),2756.1 ([M+K]^(+,) 11.8)] confirmed the structure assigned for thedesilylated tris-coupled peracylated lactose conjugate.

EXAMPLE 22

[0118] Dimethoxytrityl Tetraol

[0119] The desilylation of the tris-coupled peracylated lactoseconjugate using the above TBAF method was more problematic than for thegalactosyl conjugate probably because of the higher bulkiness and thepossible increased hindrance of the TBDPS group rendering itinaccessible to fluoride ion attack at low TBAF concentrations. Toeliminate the restrictions associated with this route another method wasused to synthesize the tris-coupled peracylated lactose conjugate.

[0120] Tetraol (prepared in Example 15) was reacted with 1.1 eq. ofdimethoxytrityl chloride (DMT-C1) in the presence of 0.1 eq. of DMAP indry pyridine at 0° C. After 10 minutes the reaction mixture was allowedto warm to room temperature. The reaction was allowed to proceed for 6hours and the crude material obtained after workup was purified bysilica gel column chromatography to give the title compound in a 64%yield.

[0121] The methylene protons at position 24 (—CH₂O—DMT) appeared as amultiplet between 2.86 and 2.94 ppm in the ¹H -NMR spectrum. The 7-and12-equatorial —CH(OH)— appeared as singlets at 3.59 and 3.76 ppm,respectively. The 3-axial —CH(OH)— appeared as a multiplet between 3.14and 3.19 ppm. The two methoxy CH₃ appeared as a sharp singlet at 3.71ppm and the phenyl protons appeared between 6.8 and 7.3 ppm. The NMRdata and the mass spectrum {FAB-NBA m/e 697 [M+H]^(+;) 303[M+H—(C₂₄H₄₁O₄)]^(+,) 100} confirmed the assigned structure.

EXAMPLE 23

[0122] Synthesis of DMT-protected Tris-NHS Cholane Derivative,DMT-protected Active Ester

[0123] Dimethoxytrityl tetraol was reacted with triphosgene (2 eq.) indry pyridine at 0° C. After 15 minutes at 0° C., N-hydroxysuccinimide(NHS, 10 eq.) was added. The reaction mixture became cloudy andthickened. Within 10 minutes the mixture started to become light red andat 15 minutes the reaction mixture was poured into cold water. The esterprecipitated out of the solution and after drying, the active ester wasobtained in 89% yield.

[0124] The 3-,7-and 12-α-CH protons appeared at 4.55-4.61 (multiplet),4.87 (singlet) and 5.03 (singlet) ppm in the ¹H-NMR spectrum. Thedownfield shift of all the α-CH and the multiplet at 2.78-2.83 ppm,corresponding to the 12 succimidyl methylene protons [—(O)C—CH₂CH₂C(O)—]confirmed the tris activation. The NMR data and the mass spectrum[(FAB-NBA) m/e: 1143.38 ([M+Na)]^(+,) 0.4), 1120.47 ([M+H]^(+,) 1.1),303.21 ([M+H—C₃₉H₅₀N₃O₄]^(1,) 100)] confirmed the assigned structure.

EXAMPLE 24

[0125] Preparation of DMT-protected Lactosylated Conjugate

[0126] The DMT-active ester was reacted with lactosylamine (3.3 eq.) indry DMF at room temperature. After 120 hours, the reaction mixture waspoured into ice water and the a 1:1 mixture of tritylated anddetritylated tris-coupled peracylated lactose conjugate was obtainedin >80% yield. The observed detritylation was not problematic since thefollowing step was the trifluoroacetic acid treatment (detritylation). Asmall amount of the mixture was subjected to column chromatography andthe two spots were separated and individually characterized by NMR andelectrospray mass spectrometry. The remainder of the mixture wascompletely detritylated in the following step.

EXAMPLE 25

[0127] Cleavage of the DMT-group with Trichloroacetic Acid (TCA)

[0128] The mixture of protected and deprotected tris substitutedperacylated lactose conjugates (Example 23) was reacted with 6% TCA indry CH₂Cl₂ at room temperature. The reaction mixture was quenched withmethanol and triethylamine after 1.5 hours. Flash column chromatographyusing a increasingly polar eluent mixture (CH₂C1₂ to 2.5% MeOH/CH₂Cl₂ to5% MeOH/ CH₂Cl₂) gave the deblocked tris peracylated lactose conjugatein 80% yield. Electrospray MS and NMR were identical to those reportedfor the tris peracylated lactose conjugate previously obtained inExample 21 using the TBAF method. Using the DMT protection scheme, 500mg of pure tris peracylated lactose conjugate was prepared as requiredfor biological testing.

EXAMPLE 26

[0129] General Method of Incorporating Tris-coupled PeracylatedGalactose Conjugate (Example 20) into an Oligonucleotide or anOligonucleoside

[0130] The tris-coupled peracylated galactose conjugate of Example 20 isactivated with disuccinimidyl carbonate (DSC, Fluka) to give thecarbamate derivative. The carbamate is then treated with4-amino-2-hydroxy butanol (prepared as per J. Am. Chem. Soc. 109, 3089.,1987) to give the corresponding carbamate. The primary alcohol functionis then treated with dimethoxytrityl chloride/pyridine to give the DMTderivative.

[0131] The DMT derivative is then phosphitylated using2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence ofdiisopropyl ethyl amine in methylene chloride as the solvent. Theresulting DMT phosphoramidite derivatized tris-coupled peracylatedgalactose conjugate is incorporated at the terminal 5′—OH or 3′—OH of anoligonucleotide or an oligonucleoside.

[0132] Alternatively the DMT phosphoramidite is coupled to a free 5′—OHposition of a nucleotide, nucleoside, oligonucleotide or anoligonucleoside. Followed by deblocking of the DMT group the freeprimary hydroxyl is available for coupling to a further nucleotide,nucleoside, oligonucleotide or an oligonucleoside to effectincorporation of the conjugate at an internal position.

EXAMPLE 27

[0133] Incorporation of Tris-coupled Peracylated Pactose Conjugate(Example 21) into an Oligonucleotide or an Oligonucleoside

[0134] Both the DMT and DMT phosphoramidite tris-coupled peracylatedlactose conjugate (Example 21) are prepared as per the procedureillustrated in Example 26. The DMT phosphoramidite is furtherincorporated onto the 3′or 5′position or at an internal position of anoligonucleotide or an oligonucleoside.

EXAMPLE 28

[0135] General Method, Attachment of the DMT-tris-coupled PeracylatedGalactose Conjugate (Example 26) onto Solid Support

[0136] DMT-tris-coupled peracylated lactose conjugate (Example 26) issuccinylated using succinic anhydride (1.5 equivalents), triethylamine(1 equivalent), 4-dimethylamino pyrimidine (0.5 equivalent) in anhydrous1,2-dichloroethane at 50° C. (as per the procedure of Kumar et al.,Nucleosides and Nucleotides, 1993, 12, 565-584). After workup theresulting succinate is dried under vacuum at room temperature. Thesuccinate is then condensed with controlled pore glass that has beenpre-acid washed (CPG Inc., New Jersey) (as per the procedure of Bayer etal., Z. Naturforsch, 1995, 50b, 1096-1100). The amino-functionalized CPGis suspended in anhydrous DMF and treated with 1 equivalent of thesuccinate, 1 equivalent of TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium tetra-fluoroborate)and 2 equivalents of N-methyl morpholine (NMM). After 12 hours ofshaking the functionalized CPG, support is filtered and washed with DMF,methylene chloride and methanol. The final rinse is carried out withether and the derivatized solid support is obtained after drying undervacuum.

EXAMPLE 29

[0137] Attachment of the DMT-tris-coupled Peracylated Lactose Conjugate(Example 27) onto Solid Support

[0138] The DMT protected tris-coupled peracylated lactose conjugate(Example 27) is attached to a solid support as per the proceduresillustrated in Example 28.

EXAMPLE 30

[0139] Preparation of Tetra-coupled Cyclohexanone PeracylatedCarbohydrate Conjugate

[0140] Cyclohexanone is treated with base (KOH) followed by fourequivalents of acrylonitrile. The resultant tetracyano compound issubjected to a Witting reaction (—C=O to —C—CH₂) followed byhydroboration to convert the olefin to a primary alcohol. Catalytichydrogenation with Pd/H₂ to convert the cyano groups to methylaminogroups will give the tetra-aminopropyl-cyclohexylhydroxymethyl compoundhaving 4 linker groups attaching 4 amino groups. Thetetra-aminopropyl-cyclohexylhydroxymethyl compound is then condensedwith carbohydrate groups using either a reductive amination route (e.g.,acylated lactose) or isocyanate condensation to give thetetra-carbohydrate substituted-aminopropyl-cyclohexylhydroxymethylcompound. The free hydroxyl group is extended with a linker such as4-amino-2-hydroxy-butanol in a similar manner as illustrated above inExample 26. Introduction of the primary and secondary hydroxyl groupsenables the preparation of the DMT or DMT phosphoramidite conjugates.The conjugates can be incorporated internally or at the 3′or 5′terminusof a nucleotide, nucleoside, oligonucleotide or an oligonucleoside asillustrated in Example 26 above.

EXAMPLE 31

[0141] Preparation of Tetra-coupled-5-aminoisophthalic Acid Conjugate

[0142] The above compound prepared as per the procedure of Hayashi etal., J. Am. Chem. Soc., 1998, 120, 4910-4915) is treated with HBr/HOACand condensed with pentaf luoro-phenyl ester of TBDPS-O-(CH₂)₆-COOH(Pg=TBDPS). The resulting tetraester is refluxed with KOH/THF/MeOH togive tetracarboxylic acid compound. Either of the carbohydratederivatives prepared in Examples 9 and 10 are condensed with thetetracarboxylic acid compound above using TBTU/NMM to give thetetra-sugar conjugated compound. The silyl protecting group is removedby treatment with tBuN⁺F and the alcohol is phosphitylated to give theconjugate phosphoramidite. The conjugate phosphoramidite is used toconjugate the carbohydrate cluster to an oligonucleotide or anoligonucleoside as illustrated in Example 26 above.

[0143] Alternatively, the free hydroxyl obtained after removal of theTBDPS group can be activated using an activating agent followed bytreatment with a linker group having one functionality to couple withthe conjugate group and also having a primary and a secondary hydroxylgroup. Introduction of the primary and secondary hydroxyl groups enablesthe preparation of the DMT or DMT phosphoramidite conjugates. Theconjugates can be incorporated internally or at the 3′or 5′terminus of anucleotide, nucleoside, oligonucleotide or an oligonucleoside asillustrated in Example 26 above.

What is claimed is:
 1. A complex comprising: a primary moiety formodulating a cellular function; and at least two cell surface receptorligands covalently connected to said primary moiety.
 2. The complex ofclaim 1 further comprising a manifold covalently connected to saidprimary moiety and each of said cell surface receptor ligands.
 3. Thecomplex of claim 2 further comprising linking moieties forming covalentlinkages from said manifold to said primary moiety and to each of saidcell surface receptor ligands.
 4. The complex of claim 1 wherein saidprimary moiety is a nucleoside, nucleotide, an oligonucleoside, anoligonucleotide or a pro-drug form of said nucleoside, nucleotide,oligonucleoside or oligonucleotide.
 5. The complex of claim 1 whereineach of said cell surface receptor ligands is the same.
 6. The complexof claim 1 comprising at least three cell surface receptor ligands. 7.The complex of claim 1 having three copies or said cell surface receptorligand.
 8. The complex of claim 1 wherein said primary compound is apeptide, protein, a molecule that can bind RNA, an antibiotic or anantibacterial compound.
 9. The complex compound of claim 2 wherein saidmanifold moiety comprises a linear chain hydrocarbon, a branched chainhydrocarbon, an aliphatic ring system, an aromatic ring system, aheterocyclic ring system, a mixed ring system having two or morealiphatic aromatic and heterocyclic rings connected or fused, whereinsaid complex compound also having a plurality of reactive sites thereon.10. The complex of claim 2 wherein said manifold is cholic acid or aderivative thereof.
 11. The complex of claim 2 wherein said cell surfacereceptor ligands are carbohydrates.
 12. The complex of claim 11 whereinsaid carbohydrates are mono-or polysaccharides.
 13. The complex of claim10 wherein said saccharides are each galactose, lactose,N-acetylgalactosamine, mannose or mannose 6-phosphate.
 14. The complexof claim 2 wherein said cell surface receptor ligands are each galactoseor lactose.
 15. A chemical complex having the formula:

wherein said primary moiety is a nucleotide, nucleoside, oligonucleotideor oligonucleoside; each of said linkers are, independently, bi-ortrifunctional; said manifold is derivatized at a plurality of sites;each of said cell surface receptor ligands is a carbohydrate; and n isan integer from 2 to about
 8. 16. The complex of claim 15 wherein saidprimary moiety is a nucleoside, nucleotide,oligonucleoside,oligonucleotide or a pro-drug form of said nucleoside,nucleotide, oligonucleoside or oligonucleotide.
 17. The complex of claim15 wherein there are at least three copies of the same cell surfacereceptor ligand.
 18. The complex of claim 15 wherein said primary moietyis a peptide, protein, a molecule that can bind RNA, an antibiotic or anantibacterial compound.
 19. The complex of claim 15 wherein saidmanifold comprises a linear chain hydrocarbon, a branched chainhydrocarbon, an aliphatic ring system, an aromatic ring system, aheterocyclic ring system, a mixed ring system having two or morealiphatic aromatic and heterocyclic rings connected or fused, whereinsaid complex compound optionally has a plurality of reactive sitesthereupon.
 20. The complex of claim 15 wherein said manifold is cholicacid or a derivative thereof.
 21. The complex of claim 15 wherein saidcell surface receptor ligands are carbohydrates.
 22. The complex ofclaim 21 wherein said carbohydrates are independently mono-orpolysaccharides.
 23. The complex of claim 22 wherein said saccharidesare, independently, galactose, lactose, N-acetylgalactosamine, mannoseor mannose 6-phosphate.
 24. The complex of claim 15 wherein said cellsurface receptor ligands are galactose or lactose.
 25. The complex ofclaim 15 wherein n is from 2 to
 6. 26. The complex of claim 15 wherein nis from 2 to
 4. 27. A method of facilitating cellular uptake of aprimary moiety comprising: identifying a cell surface receptor on atarget cell; attaching a plurality of cell surface receptor ligands onsaid primary moiety to form a complex; and contacting said cell withsaid complex.
 28. The method of claim 27 wherein said complex compoundhas the formula:

wherein said primary moiety is a nucleotide, nucleoside, oligonucleotideor oligonucleoside; each of said linkers bi-or trifunctional; saidmanifold is a multifunctional compound capable of being derivatized at aplurality of sites; each of said cell surface receptor ligands is acarbohydrate; and n is from 2 to about
 8. 29. The method of claim 27wherein said primary compound is a nucleoside, nucleotide,oligonucleoside, oligonucleotide or pro-drug form of said nucleoside,nucleotide, oligonucleoside or oligonucleotide.
 30. The method of claim27 wherein said complex comprises at least 3 copies of said cell surfacereceptor ligand.
 31. The method of claim 27 wherein said complex hasfrom three to four copies or said cell surface receptor ligand.
 32. Themethod of claim 27 wherein said primary compound is selected as apeptide, protein, a molecule that can bind RNA, an antibiotic compoundor an antibacterial compound.
 33. The method of claim 27 wherein saidmanifold comprises a linear chain hydrocarbon, a branched chainhydrocarbon, an aliphatic ring system, an aromatic ring system, aheterocyclic ring system, a mixed ring system having two or morealiphatic aromatic or a plurality of heterocyclic ring connected orfused, said complex having a plurality of reactive sites thereon. 34.The method of claim 27 wherein said manifold comprises cholic acid or aderivative thereof.
 35. The method of claim 27 wherein said cell surfacereceptor ligands are carbohydrates.
 36. The method of claim 35 whereinsaid carbohydrates are mono-or polysaccharides.
 37. The method of claim36 wherein said saccharides are galactose, lactose,N-acetylgalactosamine, mannose or mannose 6-phosphate.
 38. The method ofclaim 27 wherein said cell surface receptor ligands are galactose orlactose.
 39. The method of claim 28 wherein n is from 2 to
 6. 40. Themethod of claim 28 wherein n is from 2 to 4.